Antibodies for discrimination of prions
||Antibodies for discrimination of prions
||Zheng, et al.
||March 4, 2014
|Attorney Or Agent:
||Timian; Susan J.
|Field Of Search:
|U.S Patent Documents:
|Foreign Patent Documents:
||In the present invention, we described the use of anti-DNA antibody for the detection of prions and diagnosis of Transmissible Spongiform Encephalopathies (TSE) diseases in animals and humans.
1. A kit for the detection of infectious prions comprising: a solid support having bound thereto an anti-nucleic acid antibody, wherein the anti-nucleic acid antibody binds todeoxyribonucleic acid present in infectious prions found in Creutzfeldt-Jakob Disease brain homogenates, and a labeled prion specific antibody, wherein the labeled prion specific antibody binds to infectious prions.
2. A method for diagnosing transmissible spongiform encephalopathies in a patient comprising: drawing a sample from a patient, contacting the sample with the anti-nucleic acid antibody of the kit of claim 1 bound to the solid support, thenadding the prion specific antibody of the kit of claim 1 to form a complex between the anti-nucleic acid antibody, any infectious prions present in the sample, and the prion specific antibody, and detecting the complexes, whereby detecting the complexesprovides an indication of transmissible spongiform encephalopathies in a patient.
3. A kit for the detection of infectious prions comprising: a solid support having bound thereto a prion specific antibody, wherein the prion specific antibody binds to infectious prions, and a labeled anti-nucleic acid antibody, wherein thelabeled anti-nucleic acid antibody binds to deoxyribonucleic acid present in infectious prions found in Creutzfeldt-Jakob Disease brain homogenates.
4. A method for diagnosing transmissible spongiform encephalopathies in a patient comprising: drawing a sample from a patient, contacting the sample with the prion specific antibody of the kit of claim 1 bound to the solid support, then addingthe anti-nucleic acid antibody of the kit of claim 3 to form a complex between the prion specific antibody, any infectious prions present in the sample, and the anti-nucleic acid antibody, and detecting the complexes, whereby detecting the complexesprovides an indication of transmissible spongiform encephalopathies in a patient.
||FIELD OF THE INVENTION
In the present invention, we described the use of anti-DNA antibody (also referred to hereinafter as "an anti-nucleic acid antibody") for the detection of prions and diagnosis of Transmissible Spongiform Encephalopathies (TSE) diseases inanimals and humans.
BACKGROUND OF THE INVENTION
Transmissible spongiform encephalopathies (TSEs) comprise a group of rapidly progressing, neurodegenerative fatal diseases that affect both humans and animals. TSEs have clinical and neuropathological characteristics which include devastatingdementia, pyramidal and extrapyramidal signs with myoclonus, multifocal spongiform changes, astrogliosis, amyloid plaques, neuronal loss, absence of inflammatory reaction and are usually characterized by a long incubation period.
In animals, a commonly known example of TSE disease recognized for over 200 years, is scrapie, which is found in sheep and goats (McGowan 1922). Other animal TSE diseases have also been described, such as transmissible mink encephalopathy (TME,Marsh 1976), chronic wasting disease of mule deer and elk (CWD, Williams 1980), bovine spongiform encephalopathy (BSE, commonly known as "mad-cow" disease (Wells 1987), and the more recently described feline spongiform encephalopathy of domestic cats,pumas, and cheetahs (Wyatt 1991).
In humans, TSEs have been traditionally classified into Creutzfeldt-Jakob disease (CJD), kuru, Gerstmann-Straussler-Scheinker syndrome (GSS) and fatal familial insomnia (FFI). Among them, Kuru has been described only in the Fore linguisticgroup of New Guinea. For many years after its first recognition in 1957, kuru was the most common cause of death among women in the affected population, but its occurrence has declined because of the cessation of cannibalism that had facilitated diseasetransmission. As of today, only a few cases still occur due to the long incubation periods typical of this condition.
Although these rare neurodegenerative disorders occur in about 0.5 to one person per million worldwide (Brown 1987), TSEs attracted considerable public attention because of the unique biology and concerns about a onset of the epidemic of a newlyrecognized bovine spongiform encephalopathy (BSE) and its potential effects on human. There is mounting evidence that through dietary exposure to BSE infected tissues, it has poses a serious threat to public health and has resulted in an increasednumber of incidents of a newly recognized variant form of CJD (vCJD). Until now, there have been more than 100 cases of vCJD reported, a majority which are located in UK.
It is believed that prions are the pathogenic agent causing TSE. Many efforts have been directed towards identifying the etiological agent that causes TSEs. Early on, the transmissibility of TSE disease had been experimentally demonstrated inmany cases, kuru and CJD from humans to chimpanzees (Gajdusek 1966, Gibbs 1968), transmissible scrapie from sheep to sheep (Cuille 1936) and across species to goat (Pattison 1957). The most significant breakthrough was the successful transmission ofscrapie to mice, by Richard Chandler in 1961 (Chandler 1961). Chandler's discovery greatly facilitated TSE research by providing an experimental model that was cheaper and easier to manipulate. Although all of the above modes of transmission weredemonstrated experimentally, the cause of recent BSE in cattle and new variant CJD in human (vCJD) was considered a consequence of dietary exposure to the mix of scrapie sheep carcasses rendered for animal feed in the case of BSE (Brown 1997), and tobeef from cattle affected with BSE in the case of vCJD (Bruce 1997).
It was suggested that TSE diseases might be caused by "slow viruses" or viroids (Gajdusek 1977). However, the extreme resistance of scrapie infectivity to radiation, nucleases, and other reagents damaging to genetic materials are inconsistentwith the "virus" theory. Moreover, the infectious TSE agent could tolerate very high levels of heat and high concentrations of formaldehyde (Pattison 1965) while still able to replicate with the incubation period varying from a few months to over a year(Alper 1966).
All these "unusual" characteristics of the TSE infectious agent led Dr. Stanley Prusiner to propose the concept of "prions" in 1982 (Prusiner 1982). Prion (PrP), which stands for nucleic acid-free proteinaceous infectious particle, is aglycoprotein present in humans and animals. In humans, it is encoded by PRNP on chromosome 20 (Robakis 1986). The cellular form of this protein (PrP.sup.C) has two N-link glycosylation sites and a GPI anchor at the C-terminus. It has been mostcommonly found in neurons, and, to a much lower extent, it has also been found in other cells such as leucocytes, monocytes and platelets (Holada 2000). Furthermore, a soluble form of PrP that lacks the glycolipid anchor was detected in murine and humanserum. The transmissible scrapie disease form of the prion protein (PrP.sup.Sc) is a protease resistant isoform of its cellular precursor and is predominantly found in brain. At much lower level, it has also been found in tonsil, spleen, and lymphnodes in vCJD patients (Parizek 2001). The conversion from PrP.sup.C to PrP.sup.Sc is believed to be accomplished through a conformational change within the protein. Although there is still ambiguity concerning the mechanism of the conversion, muchexperimental evidence indicates that in the presence of PrP.sup.Sc, normal PrP.sup.C, acting as a substrate, undergoes a conformational structure change, and becomes PrP.sup.Sc. This process of propagation involves replicating the conformation ofPrP.sup.Sc in PrP.sup.C and results in PrP.sup.Sc aggregation and amyloid rod formation, hence causing cell death (Hope 1986, Horich 1997). As a result of Prusiner's concept of the "prion" as an infectious agent responsible for scrapie disease, and byextension, that of all TSE diseases gave rise to the notion of what are commonly referred to as Prion diseases to describe a class of pathologies believed to be linked to this protein.
Characteristics of PrP.sup.C and PrP.sup.Sc
The major property that differentiates PrP.sup.C and PrP.sup.Sc is their distinct conformation. The structural change from PrP.sup.C to PrP.sup.Sc is most supported by a crucial conformational change, involving a substantial increase in theamount of beta-sheet structure of the protein, with possibly a small decrease in the amount of alpha-helix, indicated by circular dichroism and infrared spectroscopy (Pan 1993, Caughey 1991). The solution structure of a fragment of the mouse PrP.sup.Chas allowed a direct determination of secondary structure content of a portion of PrP.sup.C (121-231) by NMR (Riek 1996).
Protease resistance is another characteristic that distinguishes PrP.sup.Sc from PrP.sup.C. In cultured cells and brain or in samples from many patients with GSS, PrP.sup.Sc is smaller than its cellular precursor PrP.sup.C. Even thoughcellular prion and scrapie prion are two isoform of same PRNP genomic product, PrP.sup.C is completely degraded by Proteinase K treatment while PrP.sup.Sc undergoes only limited digestion. The digestion yields a form of protein referred to as PrP 27-30in which the N-terminus has been removed. PrP 27-30 has been postulated to be the PrP.sup.Sc core required for PrP.sup.C hosted PrP.sup.Sc replication. The protease treated prion molecule, PrP 27-30 or PrP.sup.res, is tightly linked to scrapieinfectivity (Gabizon 1988), and provides additional evidence that PrP.sup.Sc is an infectious protein.
An additional attribute, perhaps linked to the significant increase in .beta.-sheet structure and concomitant protease-resistance, is the observed difference in solubility between PrP.sup.Sc and PrP.sup.C. While PrP.sup.C is a soluble protein,the PrP.sup.Sc isoform is highly insoluble. Furthermore, PrP.sup.C is found attached to the surface of neurons through a GPI tail anchored into membrane (Shyng 1994) while PrP.sup.Res is found in the cytoplasm of affected cells (Taraboulos 1990), mostlikely associated with late endosomal and lysosomal compartments (Arnold 1995), and PrP.sup.Sc is also localized in amorphous aggregates in enriched fractions from infected brain (Meyer 1986). Interestingly, a disease-associated mutant PrP, thePrP.sup.159stop mutant was found exclusively in nucleus (Lorenz 2002).
There is mounting evidence indicating a tight linkage between scrapie infectivity and PrP 27-30. Even in the purest samples, the estimated ratio of PrP molecules to infectious units is .about.10.sup.4 to 10.sup.5 (Horwich 1997, Bolton 2001). At such low levels of infectivity, it is possible that other components, co-factors, or covalent modifications, are required for infectivity. The transgenic studies on the susceptibility of mice expressing chimeric human-mouse PrP.sup.C suggest thepresence of at least one host factor other than PrP.sup.C, tentatively termed factor X, which might function as a molecular chaperone in the formation of PrP.sup.Sc (Telling 1995).
Other Molecules Associated with Prion Pathogen
About 15 to 20 strains of scrapie have been identified based on their incubation period and lesion patterns in the inbred mice. After a serially inoculation passage in inbred mice homozygous for a single PRNP genotype, all the scrapie strainsretained their original disease profile. These observations led investigators to question whether varied phenotypic strains were dominated by different conformation isoforms of same cellular prion precursor, a possibility suggested byconformation-dependent immunoassay (Safar 1998), or whether these strains were a result of various PrP.sup.Sc associated molecules.
Many researchers have identified various nonprotein molecules that are bound to prion proteins. The precise biological and physiological roles remain the topic of further investigation. Copper and zinc have been demonstrated to bind toPrP.sup.C. In vitro, these divalent metals may contribute to prion superoxide dismutase (SOD)-like activity. Such SOD-like activity and copper content are dramatically reduced in scrapie-infected brain (Wong 2001).
In addition, prion rods, composed mainly of insoluble aggregates of the N-terminally truncated prion protein (PrP 27-30) are found to be associated with 1,4-linked glucose units. Sphingolipids, polysaccharide and other membrane components werealso found in prion aggregates (Appel 1999, Klein 1998). The interaction between prion protein and lipid membranes could play a role in PrP conversion. For example, the negatively charged lipid membrane-inserted conformation of PrP is richer in.beta.-sheet structure while the binding of PrP to raft-like membranes induces the formation of .alpha.-helical structure (Sanghera 2002).
In early 1990's, Snow et al, studying Gerstmann-Straussler-Scheinker syndrome, Creutzfeldt-Jakob disease and scrapie, have documented the association of sulfated proteoglygan to the prion protein amyloid plaques (Snow 1990). In animmunohistochemistry study using heparan sulfate antibodies (anti-HS) and heparan sulfate proteoglycan antibodies (anti-HSPG), McBride has demonstrated the correlation and association between HSPG and abnormal PrP in scrapie-infected mice brain. Thiscorrelation and association was observed as early as 70 days post-infection and throughout the course of the disease (McBride 1998). In in vitro conversion from PrP.sup.C to PrP.sup.Sc and in prion infectivity reconstitution experiments, sulfate glycanshave been shown either to facilitate the conversion or to escalate infectivity (Wong 2001, Shaked 2001a). With recombinant GST::full-length prion and GST::prion fragment, Warner recently demonstrated direct binding of recombinant prion to heparin andheparan sulfate (Warner 2002). The peptide region 23-52 in prion sequence was positive in all HS and HSPG binding tests. Since the peptide failed to compete with full-length prion for binding to heparin, the author suggested that there might be anothermajor GAG-binding site in intact PrP.sup.C. Another noteworthy observation is that GAGs from different species (bovine and porcine) or from different organs (lung, kidney and intestine) have shown different affinities for prion binding. The differencein affinity may be due to prion sequence itself, or may depend on the presence of particular sugar unit in the tested GAGs.
Through a mechanism that is perhaps different from that by which glycans participate in the conversion of PrP.sup.C to PrP.sup.Sc, DNA could also convert cellular prion protein into .beta.-sheet conformation (Cordeiro 2001). Nandi demonstratedthat prion peptide 106-126 is the region that participated in the nucleic acid-prion complex association (Nandi 1998). Interestingly, not only was PK resistant amyloid aggregate obtained from the interaction between prion protein and nucleic acids, thenucleic acid morphology also changed to condensed globular structures, similar to nucleic acid structures induced by the HIV-1 NCp7 protein, but not to the structure induced by histones (Nandi 2001). Based on those in vitro conformation and conversionstudies, it was hypothesized that DNA would act as a guardian of the PrP.sup.Sc conformation as well as a catalyst to facilitate PrP.sup.Sc conversion and aggregation (Cordeiro 2001).
Whether one accepts or rejects the "protein only" or "prion only" hypothesis, the effort to link inherited information to TSE disease or the search for genetic make up related to TSE disease has never stopped. The presence of a tightly boundRNA or DNA molecule in the prion particle was proposed to explain propagation of different strains of scrapie agent with distinct phenotypes in animals homozygous for the PRNP gene (Weissmann 1991). Analysis of highly purified scrapie prions by returnrefocusing gel electrophoresis revealed the small size of remaining nucleic acids, although the size of extracted nucleotides was too small to encode any meaningful protein (Kellings 1992). In a recent report, however, Narang indicated that animalsinoculated with ssDNA purified from scrapie-hamster brains mixed with non-pathogenic prion developed clinical disease (Narang 2002). Based on his findings, he postulated that the "accessory protein" coded by the ssDNA may be involved in PrP.sup.C toPrP.sup.Sc conversion. Although the role of nucleic acids in prion-associated disease is controversial, it is clear that PrP.sup.Sc aggregates are tightly associated with these small molecules.
Infectivity and Transmissibility of Prion Diseases
Classic CJD in human has been grouped into three etiological types: sporadic (CJD), inherited (GSS or FFI), and acquired, which is very rare and includes diseases such as kuru and iatrogenic CJD. There is no hard evidence indicating any of CJDdiseases is related to animal TSEs that may have crossed species barriers. The epidemic of kuru has provided the largest body of evidence of acquired human prion disease. Searching for risk factors and possible sources of infection in sporadic CJDpatients revealed no significant correlation of disease to diet, blood transfusion or receiving other blood product. However, after intracerebral inoculation to mice, the infectivity in blood obtained from CJD patients indicated the possible presence ofthe CJD agent (Manuelidis 1985, Tateishi 1985).
BSE appears to have originated from dietary exposure. Nutritional supplements of processed meat and bone meal derived from scrapie disease infected carcasses were used to feed cattle livestock and other captive animals. In spite of BSEoriginating from scrapie, no case of de novo infection or cow-to-cow transmission has been reported.
There is mounting evidence, however, that links vCJD to BSE, The growing epidemiological data locates the majority of vCJD cases in UK where the overwhelming majority of BSE cases have also been reported. The link between vCJD and BSE isfurther supported by the neuropathologic evidence obtained from BSE-adapted macaques, the nearest model to humans (Bruce 1997), and from the study on inbred mice inoculated with the agent causing BSE and vCJD (Lasmezas 1996).
Although no vCJD patient has been documented as a victim of human-to-human transmission, the close link between BSE and vCJD attracted considerable attention. Concerns about human infection have been based on the observation that PrP.sup.Sc isreadily detectable in BSE and vCJD lymphoreticular tissues but not in classic CJD (Hill 1997), followed by the presumption that scrapie pathogen from sheep passage to cattle may have altered host range and become more adaptable to human. Experimentalprecedents for such behavior are well known: passage of mouse-adapted strains of scrapie through hamsters altered their transmissibility on back passage to mice (Kimberlin 1987, Kimberlin 1989); human strains of kuru or CJD did not transmit to ferrets orgoats until passaged through primates or cats (Gibbs 1979); and a bovine strain of BSE did not transmit to hamsters until passaged through mice (Foster 1994). Alternatively, if BSE originated from a spontaneous mutation in cattle, experimental studiesof species susceptibility to this new strain of transmissible spongiform encephalopathy (TSE) had not sufficiently advanced to predict that humans would not be susceptible.
In addition to CJD infectivity in blood described above, other TSE infectivity in blood has also been demonstrated in various experimental animals. Most blood for infectivity studies was obtained from TSE-adapted rodents such as mice andhamsters. The only exception was a study conducted in the sheep model. In this experiment, a sheep transfused with whole blood, taken from another sheep inoculated with BSE brain lysate, developed symptoms of BSE (Houston 2000, Hunter 2002). However,these experimental results yet need to be fully evaluated. The infectivity in blood has been established in rodent animals through intracerebral and intravenous transmission with mice-adapted BSE, mice-adapted vCJD and other rodent animal adapted TSEstrains. Although the infectivity in lymphocyte-rich buffy-coat is greater than in plasma, it only accounts for relatively a small portion when compared to whole blood inoculums. The molecular definition of this infectious agent present in the blood isstill under investigation. It is anticipated that finding of such infectious agent in blood would help us to better understand the relationship between PrP.sup.Sc and TSE disease.
Study on human CJD and vCJD disease indicated that genomic susceptibility may yet be another factor that may influence the spread of TSE in humans. The majority of sporadic CJD patients were found to be homozygous for Met/Met or for Val/Val atcodon 129 (Belay 1999). Nevertheless, all reported vCJD cases have been found to be homozygous for Met/Met.
The size and duration of vCJD epidemic still remains uncertain. Depending on the assumptions made and the modeling calculations employed, different predictions were proposed. One estimation of total vJCD predicts as few as 205 cases (Valleron2001). On the other hand, another prediction for vCJD mortality for the next 80 years ranges from 50 to 50,000 if infection comes only from BSE. It could reach up to 150,000 if BSE is proven to infect sheep and if subsequently it is allowed to enterhuman food chain (Ferguson 2002). Although it is impossible to make accurate predictions if the necessary parameters are either mistaken or not available, one thing is certain that if vCJD infectivity is present in blood, any prediction will be anunderestimate. In addition, vCJD has been proven to be a new disease entity and not simply the result of increased surveillance of CJD in humans (Hillier 2002).
Countermeasures have been taken by government to eliminate the spread of BSE incidence. Ruminant protein feed was banned in US and UK (1988). A series of measures have also been taken to prevent potentially infected meat from entering humanfood chain. To further reduce the human risk, FDA and CBER has issued a new policy in August 2001, which indefinitely defers any human blood donor who stayed cumulative .gtoreq.6 month during 1980-1996 in the United Kingdom (FDA 2001).
Diagnostic Assay for Prion Disease
Clinical symptoms of prion disease often overlap with those of other neuronal degenerative diseases that make diagnosis difficult. So far, PK resistant PrP 27-30 is the only protein marker linked to TSE disease. Therefore, the detection ofthis agent has become the focus of assay development. However the development of monoclonal antibody specific for PrP.sup.Sc was extremely difficult, not only because pathogenic PrP.sup.Sc isoform and normal cellular PrP.sup.C are two conformers of thesame protein with an identical primary sequence, but also because the prion appears to be a weak immunogen. The only antibody reported to be able to recognize PrP.sup.Sc specifically is not practically useful (Korth 1997). Other prion sequence-specificmonoclonal and polyclonal antibodies are unable to distinguish PrP.sup.Sc from PrP.sup.C. Nevertheless, these antibodies (such as 3F4, 6H4 described in U.S. Pat. No. 4,806,627 and EP0861900.) are still commonly in use for capture or for detection ofprion protein in combination with sample treatment and separation techniques to isolate PrP.sup.Sc from PrP.sup.C (Korth 1997, Kascsak 1987).
Since the outbreak of BSE in 1986, all commercially available tests for prion disease use, as their sample source, tissues taken from postmortem animals and humans. Among those, a tissue homogenate-based PrP.sup.Sc assay, referred to as DELFIA(dissociation-enhanced lanthanide fluoroimmunoassay), was developed for the detection of scrapie prion (Barnard 2000 and a method described in US20020137114A1). It requires a protein denaturation step using GdnHCl, in combination with optional sample PKtreatment and PrP.sup.Sc enrichment by sodium phosphotungstic acid (NaPTA) precipitation. Since the transformation of PrP.sup.C to PrP.sup.Sc is accompanied by the burial of epitopes near the N terminus of PrP, in DELFIA, monoclonal antibodies directedagainst the N-terminus of PrP are used to measure the difference of mAb binding affinity to the .alpha.-helical and .beta.-sheet conformations before and after PrP denaturation (Peretz 1997). Another conformational-dependent immunoassay (CDI) combinedwith ELISA and fluorescence detection (Safar 1998, US 20010001061 A1, US20020001817A1) was described in conformation studies in PrP.sup.Sc strains.
In a tissue distribution study of PrP.sup.Sc in vCJD patients, an improved NaPTA precipitation was described to enrich PrP.sup.Sc from brain and from other peripheral tissue homogenates (Wadsworth 2001). The modification employed endonucleasetreatment to reduce sample viscosity prior to NaPTA precipitation. The recovery of PrP.sup.Sc in the precipitated pellet was reported to be consistently greater than 90% while recovery of PrP.sup.C was about 5%. After PK digestion, the presence of PKresistant prion was verified in Western blot using 3F4 monoclonal antibody.
In another similar immunoblot assay, PK digestion was also used to eliminate PrP.sup.C. 6H4 was then used to determine the presence of PrP.sup.Sc (Schaller 1999). Based on this first generation assay, a second-generation luminescenceimmunoassay was developed in which 6H4 was coated on plates as a capture antibody. The horseradish peroxidase (POD)-conjugated detection antibody used was a mouse monoclonal anti-PrP antibody, able to form a complex with PrP27-30 bound to 6H4 (Biffiger2002).
The European Commission in 1999 evaluated 4 BSE test kits from different manufacturers (Moynagh 1999). They all used bovine brain tissue as a sample source, and all required a separate sample preparation procedure. Depending on the kitinstructions, the brain tissue homogenate needed to be processed, including denaturation, PK digestion or PrP.sup.Sc enrichment. The assay detection systems employed in DELFA, immunoblot, or in plate ELISA formats used either chemiluminescent or acolorimetric substrate.
In order to control the spread of the disease in the absence of a live-animal screening test, an extensive slaughter of cattle was carried out once an affected animal was identified within a herd. The urgency for a live animal diagnosis assaywas reinforced when the first cases of variant Creutzfeldt-Jakob disease was reported in 1996.
Antemortem TSE diagnosis development presents three major difficulties: (1) insufficient sensitivity--Except in brain tissue, PrP.sup.Sc concentrations in other tissues or fluids is considered to be very low. Therefore, a highly sensitivetechnique is required for detection. (2) Appropriate sample treatment--Any protein denaturation or PK digestion process may have a potential impact on pathogenic PrP.sup.Sc structure, with the possibility of causing a false negative result. Forexample, it has been suggested that an intermediate form of PrP.sup.Sc may not be PK resistant (Horiuchi 1999, Jackson 1999, Swietnicki 2000). And (3), the lack of PrP.sup.Sc-specific antibodies and the incompletely characterized molecular relationshipbetween the pathogenic agent and PrP.sup.Sc in blood make it difficult to design an assay format for antemortem diagnosis.
A possible approach to boost the sensitivity is in-vitro amplification of PrP.sup.Sc. It has been reported that when PrP.sup.Sc was present, repetitive cycles of sonication could induce protease-sensitive cellular PrP to form protease resistantaggregates. The authors explained that in this "protein-misfolding cyclic amplification" (PMCA) process, sonication could disrupt newly formed aggregates and generate multiple smaller units for the continued formation of new PrP.sup.Sc (Saborio 2001,WO0204954). At the end of 40 PMCA cycles, the sample was subjected to PK digestion and detected by immunoblot. It claimed that the amplification generated more than 30-fold protease resistant PrP. Since proteinase resistant PrP were generated at theexpense of the normal prion protein as substrate through amplification cycles, a large quantity of same-species normal prion was required. It has not been demonstrated whether normal prion from another species could also work as substrate, or prionprotein from a recombinant source or from sources other than brain tissue could be used. Such evidence would be useful when detection of vCJD is desired.
Immunohistochemistry of third eyelid lymphoid tissue has been described for preclinical diagnosis of ovine scrapie (O'Rourke 2000, U.S. Pat. No. 6,165,784, U.S. Pat. No. 6,261,790). Relying on a small surgical procedure, the assay makes useof sheep peripheral tissue, the third eyelid lymphoid for scrapie detection. The immunohistochemistry used a cocktail of pan-specific monoclonal antibodies to differentiate one isoform from the other. Following formalin fixation to reduce PrP.sup.Creactivity, the sample is subjected to formic acid and heat pretreatments which enhance the PrP.sup.Sc reactivity. In spite of the fact that the assay is still tissue based and the observation that PrP.sup.Sc displayed poor immunoreactivity inimmunohistochemistry staining unless treated with denaturing agents, this antemortem preclinical diagnosis has made a step towards live-animal test as well as provided a way of identification of scrapie-affected sheep during the early, preclinical stageof scrapie.
In addition to the traditional identification of pathogenic prion by eliminating cellular prion followed by non-discriminatory anti-prion antibody recognition, other reagents were found to be able to differentiate PrP.sup.Sc from PrP.sup.C, suchas plasminogen and fibrinogen. The mechanism of interaction between these human blood component proteins and PrP.sup.Sc is not clear. However, when immobilized on magnetic beads, plasminogen selectively precipitated PrP.sup.Sc from brain homogenates ofmouse, human, cattle and sheep. The evidence provided suggested that a property common to PrP.sup.Sc of various species, rather than prion primary sequence or the specific tertiary structure of individual PrP.sup.Sc molecules, could be responsible forbinding to plasminogen (Fischer 2000, Maissen 2001). The application for the use of plasminogen and other serum/plasma proteins for the capture and detection of pathogenic prion protein has been described in WO0200713 and in US20010053533A1 (Aguzzi2001).
Recent investigations have identified a new isoform of the prion protein in the urine of animals and humans with prion disease (Shaked 2001b, WO0233420A2). This isoform, referred to as UPrP.sup.Sc by the investigators, was precipitable, PKresistant, and detectable only in infected individuals but not in normal controls. Most importantly, as indicated in their publication, UPrP.sup.Sc appeared long before the clinical signs developed in inoculated hamsters. However, when UPrP.sup.Scisolated from scrapie hamster urine was inoculated back in normal hamster intracerebrally, it did not cause disease even after 270 days, well beyond the incubation period in which animal would develop clinical signs if comparable amount of brain derivedPrP.sup.Sc had been inoculated. It is not impossible that those hamsters, inoculated intracerebrally with UPrP.sup.Sc, were still in a subclinical or carrier state. Moreover, PK-resistant PrP was not found in the kidneys, which implies that thisUPrP.sup.Sc could have originated from other organs and been transported to the urine via the blood. This important observation will undoubtedly lead to a better understanding of PrP metabolism.
Therefore there remains an unmet need for a better way to detect PrP.sup.Sc and diagnose TSE in humans and animals. The aim of the present invention is to provide a non-intrusive way to isolate, concentrate and monitor the TSE disease-relatedpathogenic prion protein. The invention, including the use of selective anti-DNA antibody to bind the PrP.sup.Sc through recognition of an associated binding partner, involves the discriminatory capture of PrP.sup.Sc but not cellular prion protein. Weprovide evidence of a high affinity association of nucleic acid to PrP.sup.Sc, and we demonstrate that such nucleic acids::PrP.sup.Sc complex survived even after PK digestion and nuclease treatment.
SUMMARY OF THE INVENTION
The evidence provided in support of this invention demonstrated that PrP.sup.Sc is associated with high affinity to nucleic acid, mainly DNA as investigated. A similar association with nucleic acid was not observed with normal cellularPrP.sup.C. The evidence also demonstrated that the association was strong, resistant to PK digestion and nuclease treatment, and that PrP.sup.Sc could be readily isolated by selective anti-DNA antibodies.
This invention relates to the use of anti-DNA antibodies to capture PrP.sup.Sc through nucleic acids associated with high affinity to PrP.sup.Sc, in combination with any prion sequence-specific antibody for the detection of PrP.sup.Sc.
In another aspect, this invention relates to the selective anti-DNA antibody, as described above, that preferably binds to pathogenic prion protein but not to the normal cellular form of prion protein.
In another aspect, this invention relates to the selective anti-DNA antibody, as described above, for the detection of PrP.sup.Sc through high affinity recognition of associated nucleic acids in combination of prion sequence specific antibodies.
In another aspect, this invention relates to the selective anti-DNA antibody, as described above, for the isolation, purification, capture, elimination and monitoring PrP.sup.Sc in biological reagent production.
In another aspect, this invention relates to compositions and kits for determining the presence of PrP.sup.Sc, comprising anti-DNA antibody, as described above, for either capture or for detection step in the assay procedure.
In another aspect, this invention relates to compositions and kits for determining the presence of PrP.sup.Sc antibody produced in response to high affinity associated DNA as a binding partner to pathogenic prion protein.
In yet another aspect, this invention relates to anti-PrP.sup.Sc antibodies and their production using the said nucleic acids that can interact with and/or associate to PrP.sup.Sc, and their use in detecting nucleic acid::PrP.sup.Sc complex andprion disease infection.
In another aspect, this invention relates to a non-harsh sample treatment procedure involving nuclease digestion for the benefit of the use of selective anti-DNA antibody as described above.
Some examples of specific embodiments of the invention are as follows: A method for discriminating between infectious and noninfectious prions comprising: first contacting a sample with an anti-nucleic acid antibody, then adding a prion specificantibody to form a complex between the anti-nucleic acid antibody, prion and prion specific antibody, and detecting the complexes. A method for diagnosing transmissible spongiform encephalopathies in a patient comprising: drawing a sample from apatient, contacting a sample with an anti-nucleic acid antibody, then adding a prion specific antibody to form a complex between the anti-nucleic acid antibody, prion and prion specific antibody, and detecting the complexes, whereby detecting thecomplexes provides and indication of transmissible spongiform encephalopathies in a patient. An immunoassay for detecting infectious prions comprising: providing a solid support having bound thereto an anti-nucleic acid antibody, contacting the solidsupport with a sample, washing the support to remove any unbound sample, contacting the solid support with a prion specific antibody, and carrying out a detection step to determine if prions are bound to the solid support. A kit for the detection ofinfectious prions comprising a solid support having bound thereto an anti-nucleic acid antibody, and a labeled prion specific antibody. An immunoassay for detecting infectious prions comprising: providing a solid support coated with an agent to bind ananti-nucleic acid antibody contacting the solid support with a sample, washing the support to remove any unbound sample, contacting the solid support with a prion specific antibody, and carrying out a detection step to determine if prions are bound tothe solid support.
Another embodiment of the immunoassay described above provides a solid support coated or carrying an agent that is capable of binding the anti-nucleic acid antibody. For example, using avidin or streptavidin on the solid support andbiotinylating the anti-nucleic acid antibody so that it binds to the solid support via the avidin or streptavidin.
A further embodiment of the invention is directed to a vaccine composition comprising anti-nucleic acid antibodies and a pharmaceutically acceptable carrier. A method of treating a prion disease in a patient comprising administering atherapeutically effective amount of a vaccine composition comprising anti-nucleic acid antibodies and a pharmaceutically acceptable carrier. A method of inducing neutralized infectious prions in a patient susceptible to or suffering from a prion diseasecomprising administering a therapeutically effective amount of a vaccine composition comprising anti-nucleic acid antibodies and a pharmaceutically acceptable carrier.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1: anti-DNA IP capture and immunoblot of scrapie hamster PrP.sup.Sc. Hamster scrapie PrP.sup.Sc was immunocaptured by anti-DNA antibodies followed by SDS-PAGE and 3F4 immunoblot. Results were obtained from three separate experiments(Lane#1-4, 5-8 and 9-10).
FIG. 2: IP capture and immunoblot of BSE PrPSc. BSE PrP.sup.Sc was detected by anti-DNA antibodies and DNA binding protein through immunoprecipitation (IP) followed by 6H4 immunoblot. Results were obtained from two separate experiments(Lane#1-11, and 12-14).
FIG. 3: IP and immunoblot of hamster prion with various treatment. IP and immunoblot were performed under various conditions for each anti-DNA antibody. (1) Standard: IP performed in IP buffer with scrapie or normal hamster brain homogenate(lane 1, 5, 9, 13, 17-20). (2) DNA inhibition: 5 ug/mL phenol-chloroform extracted, ethanol precipitated, and sonicated Salmon DNA (Sigma, Mo., USA, Cat.# D7656) was added in the IP buffer as inhibitor (lane 2, 6, 10, 14). (3) Proteinase K digestion:scrapie hamster brain homogenate was treated with Proteinase K at 50 ug/mL at 37 C for 1 hour. Digestion was stopped by adding Pefablc SC to a final of 4 mM. The digested homogenate was spiked in IP buffer followed by standard IP (lane 3, 7, 11, 15). (4) Nuclease digestion: scrapie hamster brain homogenate was treated with Benzonase.RTM. nuclease at 100 U/mL at 37 C for 1 hour. Digestion was stopped by adding EDTA to a final of 10 mM. The digested homogenate was spiked in IP buffer followed bystandard IP (lane 4, 8, 12, 16).
FIG. 4. Immunocapture of PrP.sup.Sc from brains of sCJD and vCJD by OCD4. The OCD4 conjugated beads were used to immunoprecipitate PrP in clarified brain homogenates from patients affected by either sCJD or vCJD, and two unaffected subjects(normal controls). The immunoprecipitates were then analyzed by SDS-PAGE (12% gel) and Western blotting using the anti-PrP antibody 3F4. OCD4 specifically captures PrP.sup.Sc in brains of sCJD (lane 1) and vCJD (lane 3) but not PrP.sup.C in normalbrains (lanes 5 and 7). OCD4 captured PrP from sCJD and vCJD brains are authentic PrP.sup.Sc since treatment with PK (50 ug/ml for 1 h at 37.degree. C.) generates the PK-resistant core PrP.sup.res fragments (lanes 2 and 4). Right panel. The clarifiedbrain homogenates from sCJD, vCJD and normal controls were incubated in the absence (-) or presence (+) of PK (50 ug/ml for 1 h at 37.degree. C.). After the PK digestion was terminated, samples were directly loaded onto SDS-PAGE gels (12%) and analyzedon Western blots using the 3F4 antibody. Brain tissues of sCJD type 2 and vCJD are well-characterized TSE reference materials from WHO.
FIG. 5. The OCD4 based capture immunoassay for PrP.sup.Sc in brains of vCJD patients. Brain homogenates (10 ul) from 10 cases of vCJD (v1-v10) were used in the OCD4/3F4 capture immunoprecipitation assay. The experiment was conducted at theNCJDSU, UK, using kindly provided vCJD cases (case numbers: 95/052(1), 96/045(2), 97/049(3), 98/063(4), 98/148(5), 98/154(6), 99/082(7), 99/090(8), 00/066(9), 00/101(10)).
FIG. 6. Immunocapture of both the full-length PrP.sup.Sc and the PK-resistant core fragments by OCD4. Aliquots of brain homogenates from three vCJD cases were used in this experiment and conducted at NCJDSU, UK. The first aliquot was digestedwith PK (+PK). The second aliquot was digested with PK and then was subjected to immunoprecipitation by the OCD4 (+PK+OCD4). The third aliquot underwent direct immunoprecipitation by OCD4 without the PK treatment (-PK+OCD4). All above samples wereanalyzed on Western blots using the 3F4 antibody. The PK-res PrP fragments (lanes 1-5) were recovered in the OCD4 immunoprecipitates (lanes 6-10). The full-length PrP.sup.Sc in untreated samples could be efficiently captured by OCD4 as well (lanes11-15). Note that the input volume of the 10% brain homogenates was varied in some cases for better resolution of the PrP bands.
FIG. 7. OCD4 Immunocapture of PK-resistant PrP in cVJD spleen. Spleen lysate was prepared from a case of vCJD (case number 96/045 and 98/148, provided by NCJDSU at Edinburgh) using 10% homogenate followed by brief centrifuge to remove debris. PK treatment was performed at 50 ug/ml for 1 h at 37.degree. C. before the reaction was terminated by 10 mM Pefabloc. Aliquots of each 100 ul of digested spleen lysate were either pelleted by centrifugation at 14,000.times.g for 1 h at 4.degree. C.(lane 1), or were subjected to immunoprecipitation with OCD4 (lane 2 and 4) and with an unrelated mAb (lane 3 and 5, as control for non-specific binding). Detection of PK-resistant PrP in spleen was done on Western blots using the 3F4 antibody.
FIG. 8. OCD4 immunocapture of BSE PrP.sup.Sc. Brain lysates (10%, w/v) from either BSE or bovine control (CON) were prepared by homogenization in lysis buffer followed by brief centrifugation. Aliquots of clarified brain lysates (1 ul each)were used in immunoprecipitation (IP) before (lanes 1 and 2) or after (lanes 3 and 4) PK digestion (50 ug/ml for 1 h at 37.degree. C.). Additional aliquots (1 ul) without IP and PK digestion (lanes 5 and 6) served as reference for total input. Allsamples were then separated by SDS-PAGE and were detected on Western blots using the 6H4 antibody capable of recognizing bovine PrP. OCD4 immunoprecipitated PrP only from BSE brain in the absence or presence of PK. This experiment was conducted at theP3 facility of the Veterinary Laboratories Agency in Waybridge, London where brain tissues of BSE-affected and normal cattle were kindly provided.
FIG. 9. OCD4 Immunocapture of PrP.sup.Sc in scrapie sheep. Immunoprecipitation by OCD4 of PrP from brains of natural scrapie (Sc) and normal sheep control (N) before and after treatment with PK (50 ug/ml for 1 h at 37.degree. C.). Experiments were performed as described in the main text. Immunoprecipitates on the OCD4 conjugated beads were probed on Western blots with the 6H4 antibody on Western blots. Each assay used 1 ul of 10% brain homogenates.
FIG. 10. Effective immunocapture of spiked PrP.sup.Sc in human plasma by OCD4. The spike material (S) used was 1 uL of 5% homogenate of scrapie hamster brain. Standard PrP.sup.Sc immunocapture in IP buffer (1 mL) was shown in lanes 1 and 8. For lanes 2-4, the spike (1 uL) was added to 0.6 ml of three normal human plasma preparations A, B, and C (0.6 ml plasma/S) with the addition of 400 uL IP buffer. Lanes 5-7 represent the non-spiked plasma aliquots (0.6 ml plasma) with 0.4 mL IP buffer. For lanes 9-11, 5 mL each of plasma A, B, and C were preincubated with the OCD4 beads followed by brief washes of the beads in PBS. The plasma treated beads were then incubated with the spike (1 uL) in 1 mL of IP buffer (5 mL plasma.fwdarw.S). Standardimmunoprecipitation by the OCD conjugated beads was done in a total volume of 1 mL followed by Western blotting using the 3F4 antibody as described in the main text. As compared to the input control (lanes 1 and 8), significant recapture of spikedPrP.sup.Sc by OCD4 was achieved in plasma present in large excess (600 uL plasma vs. 1 uL spike) (lanes 2-4). Moreover, the fact that preincubation of OCD4 conjugated beads with large volume of normal plasma did not compromise or block its bindingability to capture PrP.sup.Sc as indicated in lane 9-11, exclude the possibility of potential OCD4 inhibitors present in human plasma. OCD4 did not capture PrP.sup.C from human plasma (lanes 5-7).
DETAILED DESCRIPTION OF THE INVENTION
The term "sample" as used herein, refers to any substance, which may contain the analyte of interest. A sample can be biological fluid, such as whole blood or whole blood components including red blood cells, white blood cells, platelets, serumand plasma, ascites, urine, cerebrospinal fluid, and other constituents of the body which may contain the analyte of interest, such as brain homogenate. Optionally, samples may be obtained from water, soil, and vegetation. The term "patient" as usedherein, refers to humans and/or animals.
Various immunoassay protocols are known and could be applied to the present invention. The assay can be carried out using any enzyme label which can be attached to the anti-prion antibody to form a labelled ligand. Enzymes such as oxidases,e.g., glucose oxidase, peroxidases, e.g., horseradish peroxidase (HRP), alkaline phosphatase and galactosidases are preferred labels. It is within the skill of one of ordinary skill in the art to determine a suitable substrate for a given label. Thesubstrate can be a material which is directly acted upon by the enzyme label or a material that is involved in a series of reactions which involve enzymatic reaction of the label. Other labels and means for detection could be for example, a ligand,nucleotide, or biotin. Detection of the labeled antibody could be by various methods including enzyme amplification with polymeric conjugates and immuno PCR.
The following examples are given to illustrate but not limit the scope of the invention.
Brain Homogenate Preparation:
Normal and scrapie hamster brain lysate were obtained from Baltimore Research and Education Foundation as 10% whole brain tissue homogenate in PBS (w/v). The lysate was further treated by adding 1/10 volume of 10.times. detergent homogenatebuffer, composed of 5% sodium deoxycolate and 5% Igpal CA-630 (equivalent to NP-40) in PBS, incubating at 4 C for 1 hr., followed by centrifugation at 1000 g for 10 minutes. The resulting supernatant was collected and used in the assay.
Normal and BSE bovine brain tissue were provided by Veterinary Laboratories Agency (VLA), UK. Normal and scrapie sheep brain tissue were provided by Animal Disease Research Unit of USDA, USA. Normal human brain tissue were provided by NationalPrion Disease Pathology Surveillance Center (NPDPSC), USA. Human sCJD and vCJD brain tissue were provided by NPDPSC and National CJD Surveillance Unit (NCJDSU), UK. Brain tissue was processed the same way (or similar) as hamster brain homogenatepreparation.
Anti-DNA Antibodies and DNA Binding Protein:
Monoclonal antibodies obtained from commercial sources were (1) murine monoclonal antibody recognizing ss-, ds-DNA, subclass IgM, Cat#12403 and subclass IgG2b, Cat#12404 from QED Bioscience, (2) murine monoclonal antibody recognizing ds-DNA,clone AE-2, subclass IgG3, Cat#2660-2308 and murine monoclonal antibody recognizing ss-, ds-, clone 49/4A1, subclass IgG2b, Cat#2660-2316 from Biogenesis. The immunogens used to raise these antibodies were Calf thymus DNA and nuclei from Raji Burkittslymphoma Cells as indicated by manufactures. Additional monoclonal antibodies from other than commercial source were also evaluated. Single Stranded Binding Protein (SSB) from E. coli purchased from Sigma (Sigma, Mo., USA, Cat.# S3917).
Preparation of Immunogens:
The immunogen used to generate anti-DNA antibodies was nuclear DNA extracted from mammalian cells based upon known protocols (Sambrook 1989) and monoclonal antibodies were also generated standard protocol (Yokoyama 2001). The antibodies werescreened by ELISA using the coated DNA immunogen.
Various cell lines are available to use in the identified protocols. For example, OCD4 and antibody AE-2 were generated from DNA extracted from Raji Burkitts lymphoma cell line. One skilled in the art would certainly recognize however thatother known cell lines and methods are available. For example, 49/4A1, 12403 and 12404 were generated from DNA extracted from calf thymus DNA and then screened by ELISA using the coated DNA immunogen.
OCD4 (100 .mu.g of purified IgG) was conjugated to 7.times.10.sup.8 tosyl activated superparamagnetic beads (Dynabeads M-280, Dynal Co.) in 1 ml of phosphate-buffered saline (PBS) at 37.degree. C. for 20 h (29). The OCD4 conjugated beads wereincubated with 0.1% bovine serum albumin (BSA) in (PBS) to block non-specific binding. The prepared OCD4 beads were stable for at least 3 months at 4.degree. C. Brain homogenate (10%, w/v) was prepared in lysis buffer (100 mM NaCl, 10 mM EDTA, 0.5%Nonidet P-40, 0.5% sodium deoxycholate, 10 mM Tris-HCl, pH 7.5, and a cocktail of protease inhibitors), followed by centrifugation at 3,000.times.g for 10 min at 4.degree. C. to remove debris. Immunoprecipitation was performed using 5 .mu.l of theclarified homogenate and 10 .mu.l of OCD4 conjugated beads in 1 ml of IP buffer containing 0.1% Tween-20 and 0.1% Nonidet P-40 in PBS, pH 7.5. After incubation with constant mixing for 2 h at room temperature, OCD4 beads were attracted to the sidewallof the plastic tubes by external magnetic force, allowing easy removal of all unbound materials in the solution. After three washes in the same buffer, OCD4 beads were collected and were boiled for 10 min in SDS sample buffer (3% sodium dodecyl sulfate(SDS), 2 mM EDTA, 10% glycerol, 50 mM Tris-HCl, pH 6.8). The eluted proteins were separated by 15% SDS-PAGE (15% Tris-glycine pre-cast gel, Bio-Rad), and were than analyzed by Western blotting either with anti-PrP antibody 3F4 recognizing residues109-112 (17) or 6H4 recognizing residues 145-152 (14). PrP bands were visualized on Kodak X-ray film using enhanced chemiluminescence.
Conjugation of Antibody and Protein to Magnetic Beads:
0.35 mL Dynabeads.RTM. M-280 Tosylactivated (Dynal Biotech, NY, USA, Cat.#142.03/04) were washed twice with PBS and the beads isolated from buffer with the magnet (Dynal Magnetic Particle Concentrator, MPC). 100 ug of purified antibody orprotein in 1 mL PBS was added to the washed beads. Incubation with rotation was performed at 37 C for 18-20 hours. The beads were isolated from the buffer with the MPC, washed twice with 1 ml PBS (0.1% BSA), and rotated for 5 minutes at roomtemperature while washing. The antibody-conjugated beads were then blocked for 3-4 hours, 37.degree. C. with 0.2 M Tris-HCl, pH 8.0, containing 0.1% BSA. The beads were subsequently washed 2 times with 1 ml PBS (0.1% BSA) and once with 1 ml PBS (0.1%BSA, 1% Tween 20) incubating each time for 10 minutes at room temp. The beads were then washed once with 1 ml PBS (0.1% BSA) and then stored in 1 ml PBS (0.05% sodium azide) at 4.degree. C.
Proteinase K Digestion and Benzonase.RTM. Nuclease Digestion:
Conditions for the PK digestion of brain lysate: Brain homogenate was suspended in PBS buffer with or without non-ionic detergent. The total homogenate protein concentration was no more than 2.5 mg/mL. PK (Roche Diagnostics, 1N, USA,Cat.#1373196) was added to a final concentration of 50 ug/mL. Incubation was at 37 C for 0.5 to 1 hour. Digestion was stopped by adding Pefabloc SC (Roche Diagnostics, 1N, USA, Cat.#1585916) to a final concentration of 4 mM.
Conditions for the Benzonase.RTM. Nuclease digestion of brain lysate: Brain homogenate was suspended in Tris-HCl buffer, with or without non-ionic detergent, containing 2 mM Mg.sup.++. Total homogenate protein concentration was no more than2.5 mg/mL. Nuclease (CN Biosciences, CA, USA, Cat.#70664) was added to a final concentration of 100 U/mL. Incubation was at 37 C for 0.5 to 1 hour. Digestion was stopped by adding EDTA to a final concentration of 10 mM.
Immunoprecipitation (IP), Non-Reducing Electrophoresis and Immunoblot Detection of PrP.sup.Sc:
Anti-DNA antibody conjugated magnetic beads were used to capture PrP.sup.Sc from brain homogenate by immunoprecipitation. The IP procedure consists of the following protocol: mix 100 uL antibody conjugated beads with 1-5 uL of brain homogenatein a total of 1 mL IP buffer (3% Tween20 and 3% Igpal CA-630 in PBS) and incubate at 25 C for 2.5 hours with rotation.fwdarw.Separate beads using MPC device and wash beads 3 times of 30 second vortexing with IP wash buffer (2% Tween20 and 2% Igpal CA-630in PBS).fwdarw.Elute captured PrP.sup.Sc by heating beads with NuPAGE sample buffer for 10-15 minutes. The eluted sample from IP capture were loaded onto a 4-12% NuPAGE.RTM. Bis-Tris Gel (Invitrogen, CA, USA, Cat.# NP0302) and subjected to non-reducingelectrophoresis at 200V for 45 minutes. The immunoblot procedure was performed as follows: transfer separated proteins in the gel to a 0.2 um PVDF membrane (Invitrogen, Cat# LC2002) at 30V for 60 minutes.fwdarw.Block the membrane with Blocker.TM. Casein in TBS (0.05% Tween20) (Pierce Chemical Corp., IL, USA, Cat.#37532) either at 25 C for 1 hour with shaking or at 4 C overnight..fwdarw.As primary antibody, use 3F4 (Signet, Mass., USA, Cat.#9620-02) at 1:3000 dilution to detect hamster and humanPrP.sup.Sc or use 6H4 (Prionics AG, Switzerland, Cat.#01-011) at 1:5000 dilution or to detect bovine and sheep PrP.sup.Sc respectively. Incubate the membrane with diluted primary antibody in 10% Blocker.TM. Casein in TBST buffer (25 mM Tri-Cl, 0.2MNaCl, 0.2% Tween20, pH 8.0) at 25 C for 1 hour with shaking..fwdarw.Wash 3.times.5 minutes with TBST buffer with shaking..fwdarw.Incubate membrane with horseradish peroxidase conjugated goat polyclonal anti-mouse IgG (H+ L) (Jackson ImmunoResearchLaboratories, PA, USA, Cat.#115-035-003) at 1:10,000 to 1:30,000 dilution in 50% Blocker.TM. Casein in TBST buffer at 25 C for 1 hour with shaking..fwdarw.Wash 6.times.5 minutes with TBST buffer with shaking..fwdarw.Add ECL chemiluminescence substrate(Amersham Biosciences, NJ, USA, Cat.# RPN2109) or SuperSignal West Dura chemiluminescence substrate (Pierce) on membrane to develop for 5 minutes..fwdarw.Take image by exposure either to Bio Max MR-2 film (Kodak, N.Y., USA) or to the ChemiDoc GelDocumentation System (Bio-Rad, CA, USA).
Vaccine and Therapeutic Uses
Another aspect of the invention is directed toward therapeutic uses of the anti-nucleic acid antibodies as a therapeutic use. Animal models can be infected, for example with vCJD. One skilled in the art would then inject the animal withanti-nucleic acid antibodies in order to bind and neutralize the infectious prions. The result would be a reduction or elimination of the disease.
The present invention uses anti-DNA to capture PrP.sup.Sc by recognition of high affinity associated nucleic acid in the nucleic acid::PrP.sup.Sc complex. Because the tight association of nucleic acid only to PrP.sup.Sc and not to PrP.sup.C,the present invention provided a non-intrusive means for the detection of PrP.sup.Sc while no PK digestion or other protein modification procedure required. It is anticipated that the mild conditions will preserve the original structure and conformationof the pathogenic prion protein, thereby offering opportunity to determine the presence of true PrP.sup.Sc while minimizing the generation of PrP.sup.Sc due to harsh sample treatment.
Provided evidence that Benzonase nuclease digestion does not compromise selective anti-DNA binding to nucleic acid::PrP.sup.Sc, including limited endonuclease treatment in sample preparation or comprised in sample buffer could eliminate theinterference of endogenous nucleic acid interference.
The use of anti-DNA antibodies offer advantages in that they display the binding specificity but can also be easily handled in direct coating to a solid phase as well as be conjugated to link to signal given reagents such as horseradishperoxidase (HRP), or to be adopted into other desired diagnosis assay format.
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